Lipid Nanoparticles–Protein Interactions: Biochemical and Biophysical Tools to Unveil the Protein Corona Biointerface and Drive Precision Delivery

Lipid nanoparticles (LNPs) represent the most clinically advanced non-viral nucleic acid delivery systems; yet, the protein corona (PC) that spontaneously assembles on their surface in biological fluids largely dictates their biodistribution, rendering the LNP–PC biointerface a key determinant in achieving the more efficient and selective delivery to non-hepatic organs. Conventional endpoint analyses fail to resolve the kinetic and structural complexity of the LNP–PC biointerface. This review provides a systematic overview of the methods for studying this biointerface, covering the isolation of LNP–PC complexes, such as differential centrifugation (DC), density gradient centrifugation (DGC), magnetic separation, photoaffinity separation, antibody-based pulldowns, size exclusion chromatography (SEC), and asymmetric flow field-flow fractionation (AF4); the characterization of PC properties, such as dynamic light scattering (DLS), multi-angle light scattering (MALS), nanoparticle tracking analysis (NTA), electrophoretic light scattering (ELS), transmission electron microscopy (TEM), nuclear magnetic resonance spectroscopy (NMR), fluorescence correlation spectroscopy (FCS), liquid chromatography-mass spectrometry (LC-MS), circular dichroism (CD), and analytical ultracentrifugation (AUC); the real-time monitoring of binding kinetics using quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and biolayer interferometry (BLI); the characterization of binding affinity by isothermal titration calorimetry (ITC), microscale thermophoresis (MST), and spectral shift (SpS); and high-throughput screening methods for LNPs. Integrating these tools permits the discrimination of hard corona (HC) and soft corona (SC) populations, the quantitative determination of adsorption kinetics and binding affinity, and the correlation of PC compositional fingerprints with LNP tropism. This integrated workflow transforms the LNP–PC biointerface from an ill-defined biological layer into a programmable information interface, offering a methodological guide to accelerate the rational design of next-generation LNPs.